1. Field of the Invention
The present invention relates generally to solid phases for use in connection with biological assays. More specifically, the invention pertains to solid materials which can be used for chemiluminescent assays based on 1,2-dioxetanes which can be triggered by enzymes and enzyme conjugates in ligand-binding pairs to emit light.
2. Background of the Technology
Increasingly, blotting assays employing chemiluminescent detection have become a popular modality for the detection of biomolecules such as proteins and nucleic acids. Conventionally, such assays are conducted by isolating a sample of the analyte on a membrane, exposing that membrane to an antibody-agent or nucleic acid probe agent complex, wherein the agent causes a composition to be added to undergo chemiluminescence. In its most widely practiced form, this type of blotting assay employs an enzyme or enzyme conjugate as the agent causing a compound which can decompose to release light to undergo that decomposition, thereby giving chemiluminescence. Among the most effective compounds for this purpose are 1,2-dioxetanes. These structures, if stabilized with an adamantyl group or similar stabilizing group, or with a derivatized adamantyl group, can be protected with an enzyme-labile group to form an enzyme cleavable substrate. When cleaved by a suitable enzyme, the substrate forms an unstable anion, which then decomposes to release light. For example, using an enzyme, the chemiluminescent compound is a substrate, and among dioxetane substrates, AMPPD disodium 3-(4-methoxy-spiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.13,7]decan]-4-yl) phenyl phosphate is widely used. Structurally related compounds, wherein the adamantyl group is substituted with various electron-active groups, convert the adamantyl moiety from a mere stabilizing agent to one which actively influences dioxetane decomposition. Among these, the chlorine-substituted compound, or CSPD, has been demonstrated to be particularly effective. A wide variety of other compounds, bearing other enzyme-labile protective groups, such as sugar, acetate and other ether and ester moieties are known and effective.
Various protocols are used in blotting assays. In a Western or Southern Blotting assay, for example, proteins or nucleic acids are purified and transferred to membrane supports. The membranes are typically nitrocellulose, polyamide (e.g. nylon), or polyvinylidene fluoride (PVDF). This transferred material (analyte) is incubated with at least one antibody specific for the compound being sought (e.g., specific protein or nucleic acid). In a Western Blotting assay, the antibody can be complexed with an enzyme, or, alternatively, a second antibody complexed with an enzyme can be added following a washing step. In the case of AMPPD and CSPD, the binder (antibody or DNA/RNA probe) is conjugated with an alkaline phosphatase enzyme. Subsequent to washing, the blot is incubated with the chemiluminescent substrate. Release of chemiluminescence is confirmation of the presence of the suspected compound or target analyte.
In Southern blotting procedures, a nucleic acid sample is blotted onto a membrane following gel electrophoresis separation. Hybridizations are performed with enzyme labeled nucleic acid probes (labeled directly or indirectly via biotin-avidin or antibody-antigen bridge) containing base sequence complementary to regions specific for the target sample. Again, subsequent to washing, the blot is incubated with the chemiluminescent substrate and the subsequent release of light signal provides confirmation of the presence of the suspected nucleic acid sequence.
These blotting formats present certain problems in connection with the membrane supports typically employed. For instance, the chemical content of the membrane surface, to which the chemiluminescent substrate is exposed, has a tendency to quench or promote quenching of the emitted light, thus reducing the intensity of the chemiluminescent signal. Further, the membranes used have significant lot-to-lot variations, due to current production processes. As a result, it is difficult both to standardize the process and to provide for automatic data acquisition. Among specific problems encountered are low signal levels, very high non-specific backgrounds, and membrane-initiated decomposition of chemiluminescent substrates, such as AMPPD and CSPD.
When dealing with dioxetane substrates such as AMPPD, it is important to note that these compounds have very low intensities of chemiluminescence in aqueous or protic environments. This is believed to be due principally to proton transfer quenching reactions, or dipole-dipole interactions which tend to promote dark reactions of the excited state ultimately produced by enzyme cleavage. Proton transfer reactions are extremely well known in organic chemistry, and can easily compete with light emission during the lifetime of the excited state, which is several orders of magnitude longer. See, for example, Shizuka, “Accounts of Chemical Research”, 18, 141-147 (1985). This can be confirmed by the fact that the chemiluminescent efficiency of AMPPD in aqueous buffers is approximately only 10−6, but improves, in the presence of a hydrophobic medium, by approximately 104.
In addition to the above-noted problems, conventional blotting assays continue to leave certain goals unmet. Of particular importance is the inability to quantify the amounts of individual nucleic acid fragments, or proteins, identified in blotting applications. Currently, blotting assays are qualitative in nature, confirming only the presence or absence of a particular component (nucleic acid or protein). Frequently, a component will be present to varying extents in multiple analytes, but diagnosis of a disease or particular molecular biological condition depends on the relative number of component species in the analyte. Current blotting techniques do not permit adequate quantitative discrimination on this basis.
Another shortcoming of prior art blotting assays is the resolution obtainable with conventional solid support materials. It would be desirable to provide material compositions which would enable the quantitative detection of sharply resolved signal generating regions, corresponding to a bound component in the absence of or greatly diminished greatly diminished background signal. Such a material compositions would be suitable for automated data acquisition. As an example, scanning charged-coupled devices can be employed in reading complex information such as spatially localized DNA or RNA sequences on a surface. Such automation would enable a higher efficiency of error-free data acquisition. Current blotting assays conducted on surfaces utilizing chemiluminescent compounds such as dioxetanes do not provide the necessary sharp resolution of high intensity signal producing regions or loci with sufficiently diminished background to permit automated data acquisition. More importantly, these blotting methods and materials are wholly insufficient for application to high-density molecular (nucleic acid or protein) microarrays where more quantitative individual target quantitation is desirable.
Various solutions to the aforementioned problems have been proposed. For example, cationic layers comprising onium salt containing polymers have been proposed for use as membranes in chemiluminescence assays. These membranes have been used to reduce the background noise and to improve the sensitivity and reliability of chemiluminescent assays. See, for example, U.S. Pat. Nos. 5,336,596; 5,593,828; 5,827,650 and 5,849,495.
A need still exists, however, for solid phases which can be used for chemiluminescent assays based on enzyme-triggerable dioxetanes which yield improved signal collection from higher feature density signal generating regions. These solid phases would enable a more quantitative analysis of spatially localized analytes on/in the surface of the material. Such materials would allow for improved chemiluminescent signal detection particularly in high density microarray analysis formats.